The present invention, in some embodiments thereof, relates to methods of increasing metabolic maturation of immature hepatocytes and isolated hepatocytes resulting thereof.
The liver is the largest internal organ in the human body, and is responsible for protein synthesis as well as glucose, lipid and nitrogen homeostasis. Transformation of lipid metabolites is primarily carried out by the cytochrome P450 (CYP450) family of monooxygenases, which is also responsible for the transformation of most xenobiotics, often as a first step for conjugation and secretion of water-soluble metabolites (Guengerich 2007). Due to these metabolic functions the organ is particularly sensitive to drug-induced liver injury (DILI), a leading cause of acute liver failure and post-market drug withdrawals (Kaplowitz 2005). Liver toxicity and drug metabolism are therefore a major focus of pharmaceutical and cosmetic industry compound development.
The low concordance between animal studies and clinical data (Olson, Betton et al. 2000, Gottmann, Kramer et al. 2001) and the low metabolic activity of hepatic cell lines necessitates the use of primary human hepatocytes for drug metabolism and toxicity studies (LeCluyse 2001). However, primary human hepatocytes are scarce, do not proliferate and rapidly lose their metabolic functions in vitro (Guillouzo 1998, Hewitt, Lechon et al. 2007). The recent development of micro-fabricated or oxygenated co-cultures was shown to support primary cell activity for several weeks in culture (Nahmias, Berthiaume et al. 2007, Khetani and Bhatia 2008, Kidambi, Yarmush et al. 2009, Shulman and Nahmias 2013), but did little to attenuate the need for functional cells. It is the scarcity in primary human hepatocytes that drives the current focus in hepatic cell differentiation. Although a few cell types can be coaxed into hepatic like cells (Schwartz, Reyes et al. 2002, Lue, Lin et al. 2010, Stock, Bruckner et al. 2010, Zhu, Rezvani et al. 2014), it is thought that only pluripotent stem cells (PSC) may provide the full gamete of mature hepatic function (Duan, Ma et al. 2010).
Indeed, several groups already reported the differentiation of hepatocyte-like cells from embryonic or induced pluripotent stem cells (Song, Cal et al. 2009, Duan, Ma et al. 2010, Si-Tayeb, Noto et al. 2010, Chen, Tseng et al. 2012, Roelandt, Vanhove et al. 2013, Shan et al. 2013, Chen et al. 2015). While these groups focused on albumin production, hepatocyte-like cells still display fetal markers such as α-fetoprotein (AFP) and lack the inducibility and function of most mature CYP450 enzymes, such as CYP3A4. In fact, recent attempts to use hPSC-derived hepatocytes in drug toxicity screening garnered a poor correlation with primary human hepatocytes, showing an R2 of 0.49 (Szkolnicka, Farnworth et al. 2014). Interestingly, fetal markers such as AFP and CYP3A7 were shown to decrease only after birth, with a gradual increase in CYP3A4 expression taking place only during the first year of life (Lacroix, Sonnier et al. 1997, Guengerich 2007). These in vivo results suggest that post-partum cues may drive the final maturation step of liver cells.
Postnatal maturation of mitochondria is another key limiting factor in the derivation of functional hepatocytes. Fetal hepatocytes rely on placenta-transferred carbohydrates and anaerobic glycolysis (Hommes, 1973; Hommes, 1975), while postnatal functional and structural maturation of over 1400 mitochondria in liver cells enables much higher metabolic rates (Pollak, 1980).
Therefore, recently, mitochondrial biogenesis and metabolism emerged as important factors in evaluating hepatic maturity and functionality in vitro (Yue Yu, 2012; Anais Waneta, 2014).
The liver microenvironment changes significantly by the transition from placental to enteral nutrition (Morelli 2008). Fatty acids from breastfeeding become the primary energy source, while gut colonization exposes the liver to bacterial-derived secondary metabolites, such as litocholic acid (LCA) and menaquinone-4 (MK4). LCA is a secondary bile acid, produced by intestinal bacteria, and shown to activate the pregnane X receptor (PXR), a nuclear receptor controlling the expression of CYP450 enzymes such as CYP2C9 and CYP3A4 (Staudinger, Goodwin et al. 2001). Vitamin K is a group of essential fat-soluble vitamins, whose active metabolite MK4 (vitamin K2) is synthesized by colon bacteria (Conly and Stein 1992).
Prenatal levels of vitamin K are low due to poor placental travel (Shearer, Rahim et al. 1982), and it is regularly administered to newborns immediately after birth to prevent vitamin K deficiency that leads to fatal bleeding (Shearer 2009). MK4 was also shown to activate PXR, primarily in bone cells (Tabb, Sun et al. 2003, Ichikawa, Horie-Inoue et al. 2006).
Intestinal microbial colonization in newborns is also influenced by the lipid rich diet (Morelli, 2008; DA, 2014). Bifidobacterium and lactobacillus thrive on breast milk glycans and lactate, respectively, thus becoming predominant during the lactation period (Conway, 1997; Haarman, 2005; Haarman, 2006; Sela, 2014). Both strains metabolize one of the main unsaturated fatty acid in the human breast milk, linoleic acid (LA) (Finley, 1985, Supplement table 1), to conjugated linoleic acid (CLA), mainly to cis-9,trans-11-octadecadienoic acid 18:2 (9CLA), which is known for its bioactive properties (Halade, 2009; Halade, 2010; Poirier, 2006; Reynolds, 2010; Choi, 2007). 9CLA enhances hepatic mitochondrial function in rats (Choi, 2007) and acts as a high affinity ligand of Peroxisome proliferator-activated receptor, isoform a (PPARα) (Moya-Camarena, 1999). PPARα is a lipid activated nuclear receptor whose expression and activity increase significantly during the suckling period (Beck, 1992; Panadero, 2000).
Additional background art includes U.S. Patent Application Publication US 20070213282 A1 [Peroxisome proliferator-activated receptor (PPAR) activator, and drugs, supplements, functional foods and food additives using the same]; Tashiro K., et al., 2009 (Stem Cells 27: 1802-1811); Inamura M et al. 2011 (Mol. Therapy, 19:400-407); Sullivan G J., et al. 2011 (Hepatology 51: 329-335); Si-Tayeb K., et al., 2010 (Hepatology 51: 297-305); Song Z., et al. 2009 (Cell Research 19: 1233-1242); Shan J., et al., 2013 (Nature Chemical Biology 9: 514-521); Kai-Ting Chen et al., 2014 (Journal of Hepatology, Elsevier, 2014, 61 (6), pp. 1276-1286); Parmentier J H 1997 (Biochemical Pharmacology 54: 889-898); Gruppuso P A., et al. 2000 (Biochimica et Biophysica Acta 1494:242-247); Esmaeli S., et al. 2014 (Cell Biochemistry and Function 32: 410-419); Chen J., et al., 2016 (Scientific Reports 6: 18841 DOI: 10.1038/srep18841); Stier H., et al., 1998 (Differentiation 64: 55-66).